The invention described herein relates generally to the field of human disease, and more specifically to treating and diagnosing disease based on nucleic acid sequences that encode a human Cell Growth Regulatory Protein(s) that regulates cell growth by Phosphorylation of cell cycle proteins.
Oncogenic transformation of cells leads to a number of changes in cellular metabolism, physiology, and morphology. One characteristic alteration of oncogenically transformed cells is a loss of responsiveness to constraints on cell proliferation and differentiation normally imposed by the appropriate expression of cell growth regulatory genes. While different types of genetic alterations may all lead to altered expression or function of cell-growth regulatory genes and to abnormal growth, it is generally believed that more than one event is required to lead to neoplastic transformation of a normal cell to a malignant one (Land et al. (1983) Nature 304: 596; Weinberg R A (1989) Cancer Res. 49: 3713). The precise molecular pathways and secondary changes leading to malignant transformation for most cell types are not clear. A number of cases have been reported in which altered expression or activity of some proteins with putative cell-cycle control functions and/or implicated in the formation of functional transcriptional complexes, such as p53 and RB, can lead to loss of proliferation control in cells (Ullrich et al. (1992) J. Biol. Chem. 267: 15259; Hollstein et al. (1991) Science 253: 49; Sager R (1992) Curr. Opin. Cell. Biol. 4: 155; Levine et al. (1991) Nature 351: 453).
Regarding cell cycle aspects of oncogenic transformation, several proteins involved in regulating the cell cycle have recently been identified and shown to be critical regulators of mitosis. The progression of a eukaryotic cell through the cell cycle to mitosis involves a number of what appear to be tightly regulated mutually antagonistic phospatases and kinases. A key kinase this process is cdc2, which is thought to be responsible for initiating mitosis.
Cdc2 is involved in a number of downstream mitotic events including metaphase alignment of chromosomes, segregation of sister chromatids in anaphase, and cleavage furrow formation. A large number of proteins involved in these processes are phosphorylated by cdc2 including histones, lamins and microtubule-associated proteins. See, Nigg, Semin. Cell Biol. 2: 261-270 (1991). Particularly noteworthy is the phosphorylation of caldesmon, an actin-associated protein, which is thought to be necessary for the dissolution of M-phase specific actin cables. Well known changes in nuclear structure also involve cdc2. For instance chromatin condensation involves cdc2 phosphorylation of the histone H1. See, Langan et al. Molec. Cell. Biol. vol. 9: 3860-3868. The dissolution of the nuclear envelope is associated with cdc2 phosphorylation of lamin B. See, Peter, et al. Cell, vol. 61: 591-602 (1990). Further, during mitosis the nucleolus disappears and this event is also associated with cdc2 activity.
Considering the importance of cdc2 to critical cell cycle functions, and therefore to cell growth, one would expect that this enzyme is subject to several significant control mechanisms. Indeed two such mechanisms have been identified and include the phosphorylation of cdc2 at three sites; tyrosine 15, threonine 14 and threonine 161. Phosphorylation of threonine 161 and dephosphorylation of tyrosine 15 and threonine 14 are necessary for kinase activity. The phosphorylation of tyrosine 15 and threonine 14 is thus a negatively regulator of cdc2 kinase activity. See, Mueller, P. R., et al. Science, vol. 270, pages 86-90 (1995). An enzyme termed cyclin-dependent kinase (CDK)-activating kinase (CAK) phosphorylates cdc2 on threonine 161. An enzyme termed Wee1 kinase has been shown to phosphorylate cdc2 at tyrosine 15 but not threonine 14. See, Mueller, P. R., et al. Mol. Biol. Cell vol. 6 page 119 (1995). Recently, a second enzyme has been identified from Xenopus that phosphorylates both amino acids, and has been termed Myt1 for its membrane association, and its capacity to phosphorylate both tyrosine 15 and threonine 14 on cdc2. There is significant nucleotide homology between the Weel and Xenopus Myt1 enzymes.
It is worth noting certain other proteins involved in cdc2 action. In order for cdc2 to exhibit kinase activity, and thus phosphorylate its mitotic substrates the phosphates at positions tyrosine 15 and threonine 14 must be removed. This is realized by the phosphatase cdc25. Cdc25 is, in turn, activated by cdc2 phosphorylation. See, Hoffmann, et al., EMBO J. vol: 12; page 53 (1993). Cdc2 is part of a complex consisting of a cyclin, cyclin B. The phosphorylation events that occur on cdc2 occur after its association with cyclin B.
The identification and isolation of a human enzyme with kinase activity similar to Xenopus Myt1 will have significant medical applications both as an inhibitor of uncontrolled cell growth as displayed by cancer cells, and as a means to identify small molecules with similar activity in drug screening assays. Moreover, the enzyme will have diagnostic applications.
A first object of the invention is to describe a family of related isolated nucleic acid sequences that encode proteins with kinase activity, hereinafter referred to as Cell Growth Regulatory Proteins.
A second object of the invention is to describe a family of related isolated nucleic acid sequences that encode such Cell Growth Regulatory Proteins having molecular weights ranging from about 45-60 kdaltons.
A third object of the invention is to describe an isolated human Cell Growth Regulatory Protein having a calculated molecular weight of about 54.6 kdaltons that phosphorylates Cdc2 of the Cdc2/CyclinB complex at amino acid residues threonine 14 and tyrosine 15, which phosphorylation substantially inhibits the kinase activity of the Cdc2/CyclinB complex.
A fourth object of the invention is to describe isolated nucleic acid sequences that encode protein fragments, or the fragments themselves of Cell Growth Regulatory Protein(s), respectively.
A fifth object of the invention is to describe host cells transformed with isolated nucleic acid sequences that encodes Cell Growth Regulatory Protein(s) or fragments thereof.
A sixth object of the invention is to describe vectors that contain isolated nucleic sequences that encode Cell Growth Regulatory Protein(s) or fragments thereof.
A seventh object of the invention is to describe complexes consisting of full length or fragments of Cell Growth Regulatory Proteins and cell cycle target proteins.
An eighth object of the invention is to describe methods of diagnosing disease, preferably those involving unwanted cell growth, including cancer, using isolated nucleic acid sequences, or fragments thereof, that encode a Cell Growth Regulatory Protein, or fragments thereof.
A ninth object of the invention is a description of antibody that binds to Growth Regulatory Protein, or fragments thereof.
A tenth object of the invention is to describe an assay using isolated nucleic acid sequences that encode a Cell Growth Regulatory Protein, or fragments thereof for identifying compounds that would have therapeutic applications for the treatment of diseases involving unwanted cell growth, including cancer.
These and other objects of the present invention will become apparent to one of ordinary skill in the art upon reading the description of the various aspects of the invention in the following specification. The foregoing and other aspects of the present invention are explained in greater detail in the drawings, detailed description, and examples set forth below.